Papillomaviruses are DNA viruses that infect the skin and mucous membranes of humans and animals. Approximately 130 types of human papillomaviruses (HPV) have been identified, of which between 30-40 types are transmitted through sexual contact and infect the anogenital region. Some of these HPV types cause genital warts, while others do not cause any noticeable signs of infection. At least 14 HPV types have been associated with a high risk for cervical cancer, namely types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68. Detection of these high risk types of HPV is important in the prevention of cervical cancer.
The genome of all HPV types is similarly organized. A number of early (E) and late (L) proteins are specifically encoded. E1 and E2 proteins are required for DNA replication. E4 and E5 proteins are required for replication of the viral genome in the upper layers of the epithelium. E6 and E7 proteins are oncogenic and cooperate to immortalize cells and to induce genomic instability. L1 and L2 proteins form the viral capsid and are expressed late in infection in the upper layers of the epithelium. Another part of the genome, namely the long-control-region (LCR), contains most of the regulatory DNA sequences needed for proper replication of the viral genome and expression of the viral genes.
A variety of methods for detecting high risk types of HPV have been devised. Many rely on the detection of unique sequences in the HPV genome. For example, DNA or RNA probes complementary to a portion of the genes of HPV type 35, have been described, such as in U.S. Pat. No. 4,849,332, as useful in screening for the presence of this type of HPV in test samples. Additional probe sequences useful for detecting oncogenic HPV types are disclosed in U.S. Pat. No. 6,265,154. U.S. Pat. No. 5,705,627 teaches the use of polymerase chain reaction (PCR) to amplify and detect HPV DNA using degenerate or mixed consensus primers, followed by typing using a mixture of genotype-specific DNA probes. Other examples of using consensus primers can be found in U.S. Pat. No. 5,364,758 and Kleter, B. et al., Am. J. of Pathology, 1998, 153(6):1731-39. Moreover, many of the methods known in the art also involve detecting human beta globin sequences in test samples.
As illustrated above, a variety of methods for detecting high risk types of HPV are known in the art. Despite such methods, there exists a need in the art for new methods that: (1) are capable of detecting multiple HPV genotypes in a single reaction while at the same time differentiating the detection of certain specific genotypes from others (e.g., partial genotyping); (2) do not exhibit any cross-reactivity between HPV types; (3) provide a robust clinical sensitivity and specificity; and (4) provide high throughput and efficient workflow.